Phagocytic white blood cells and antimicrobial agents have been recognized as having several potential interactions that may be synergistic for combating infection. Phagocytic killing by polymorphonuclear leukocytes (PMNs), monocytes, and macrophages is the primary host defense against bacterial infections. Antimicrobial agents make bacteria more susceptible to killing by neutrophils even at subinhibitory concentrations (Adinolfi & Bonventre (1988) Antimicrob Agents Chemother 32: 1012-8). Neutrophils migrate to sites of infection, concentrate at these sites, and thus may serve as an antimicrobial agent delivery mechanism.
Despite the effectiveness of this defense, Salmonella and other intracellular pathogens can invade phagocytes and survive inside them, avoiding the lysosomal compartment. Cellular invasion is an important step in the progression of many serious bacterial infections because it allows pathogens to evade host defense mechanisms and benefit from a rich nutrient supply.
In order for neutrophils to function as an effective means of transporting antimicrobial agents to sites of infection, several criteria must be met: the agent should not interfere with neutrophil migration, the agent should be concentrated in the neutrophil, and the agent should be released in an active form at the site of infection.
Cell-permeating antimicrobial agents can potentially play an important role in eliminating infections by intracellular pathogens. Unfortunately, many antibiotic classes do not penetrate the plasma membrane effectively (See, for example, Table 1). β-lactam and cephalosporins, while representing one of the most prescribed antibiotics today, suffer from poor intracellular penetration and therefore have limited utility in the treatment of intracellular bacterial pathogens. Therefore, the effectiveness of an antimicrobial agent in vivo depends not only on its activity but also on its ability to reach sites of infection.
TABLE 1Antimicrobial agent uptake by PMNaConcn (mg/ml)AgentExtracellularIntracellularI/E ratioAzithromycin0.151.7 ± 4.3517Ciprofloxacin4.024.8 ± 4.86.2Levofloxacin6.028.8 ± 4.84.8Moxifloxacin4.5  54 ± 10.512.0Penicillin G10 1.6 ± 0.20.16Telithromycin0.119.7 ± 2.7197aExtracellular and intracellular antimicrobial agent concentrations for PMN incubated with the indicated concentrations of antimicrobial agents for 1 h were determined by bioassay. Results are means of at least three determinations.
One class of antibiotics which have shown promise both in terms of accumulation in phagocytic white blood cells as well as in fighting intracellular infections has been macrolides. Certain members of the macrolide antibiotic family accumulate to a large degree in phagocytic cells, often achieving a cellular-to-extracellular concentration ratio (C/E) of greater than 100. In particular, Azithromycin, an azalide antibiotic (see, e.g., Djokic et al. (1988) J. Chem. Res. 152: 1239-61; Bright et al. (1988) J. Antibiot. 41: 1029-47; and U.S. Pat. No. 4,474,768) is more stable than erythromycin in the presence of acids, and has very low plasma concentrations owing to its concentration to a large extent in cells, often achieving a C/E of ˜500 (Bouvier d'Yvoire et al. (1998) J. Antimicrob. Chemother. 41: Suppl. B, 63-68). Its stability, accumulation in phagocytes and long half-life (t1/2˜68 hours), make azithromycin an ideal antibiotic in terms of in vivo distribution.
In spite of the ideal intracellular distribution of macrolide antibiotics, resistance is emerging among bacterial pathogens (see, e.g., Singh et al. (2001) Antimicrob Agents Chemother 45: 263-266; Occhialini et al. (1997) Antimicrob Agents Chemother 47: 2724-2728; and Nash (2001) Antimicrob. Agents Chemother. 45: 1607-1614). It is clear that development of more intracellularly accumulating antibiotics, whether new or existing, will greatly enhance treatment of various infectious diseases.
In general, successful therapy with a pharmaceutical agent requires that the agent satisfy numerous requirements imposed by the physiology of the host and of the disease or condition. These include: (i) adequate ability to interact with the target; (ii) appropriate physical properties for presence at the location of the receptors in concentrations that permit the interactions noted above; (iii) appropriate physical properties to allow the agent to enter the body and distribute to the location of the receptors by any means; (iv) Sufficient stability in fluids of the body; (v) the absence of toxic effects in compartments where the drug is most concentrated, or in any other compartment where the drug is located; and (vi) the absence of sequestration into non-physiological compartments and so on.
In general, these compounding requirements limit the nature of pharmaceutical compounds that have utility in vivo and thus reduce the probability of discovering adequately active molecules from de novo starting points.
Current strategies for enhancing the intracellular accumulation of antibiotics include direct chemical modification of regions within the antibiotic, incorporation of antibiotics into liposomes, or the preparation of prodrugs. Recent improvements in the technology of synthetic chemistry and molecular biology have allowed the testing of large numbers of structural variants and the discovery of many ligands with adequate affinity to their targets to have some potential in vivo. Many such molecules prove inadequate on in vivo testing largely due to the manifold, stringent, and often conflicting (i.e., stability without toxicity) requirements outlined above.
U.S. Pat. No. 5,434,147 describes a process for conjugation of antibiotics with transferrin or low density lipoprotein for treating intracellular pathogens. The coupled transferrin molecules are claimed to be selectively taken up by phagosomes to target membrane-bound pathogens. However, transferrin, and therefore molecules attached to it, do not traffic through the lysosomal compartment. This process, therefore, is of limited utility in areas where the antibiotic is to provide synergistic activity with phagocytic immune cells in neutralising non-intracellular pathogens. Furthermore, the molecular weight of transferrin (76,000-81,000 daltons), as well as its polypeptide composition, precludes oral delivery of such compositions. Oral absorption of drugs is the most desirable method of drug administration in the treatment of human diseases, particularly in prolonged therapeutical treatments.
One class of antibiotics which have shown promise both in terms of accumulation in phagocytic white blood cells as well as in fighting intracellular infections has been macrolides. Certain members of the macrolide antibiotic family accumulate to a large degree in phagocytic cells, often achieving a cellular-to-extracellular concentration ratio (C/E) of greater than 100. In particular, Azithromycin, an azalide antibiotic (Djokic et al., supra; Bright et al., supra; and U.S. Pat. No. 4,474,768) is more stable than erythromycin in the presence of acids, and has very low plasma concentrations owing to its concentration to a large extent in cells, often achieving a C/E of ˜500 (Bouvier d'Yvoire et al., supra). Its stability, accumulation in phagocytes and long half-life (t½˜68 hours), make azithromycin an ideal antibiotic in terms of in vivo distribution.